1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of bacteria and eucarya. More specifically, this invention relates to novel PNA probes, probe sets, methods and kits which can be used to detect, identify or quantitate one or more bacteria and/or eucarya which may be present in a sample.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, fungi, virus or other organisms and are also useful in examining genetically-based disease states or clinical conditions of interest. Nonetheless, probe-based assays have been slow to achieve commercial success. This lack of commercial success is, at least partially, the result of difficulties associated with specificity, sensitivity and reliability.
Hybridization assays hold promise as a means to screen large numbers of samples for conditions of interest. In practice, however, it is often difficult to multiplex a hybridization assay given the requirement that each of the many very different probes in the assay must exhibit a very high degree of specificity for a specific target nudeic acid under the same or similar conditions of stringency. Given the difficulties in specificity, sensitivity and reliability of nucleic acid probes in assays designed to detect a single target nucleic acid, sensitive and reliable methods for the multiplex analysis of samples have been particularly elusive.
The in-situ targeting of rRNA as a means to detect, identify or quantitate organisms is well established (See: Amann et al., Microbiological Reviews, 59:143-169 (1995). Nucleic acid probes for the universal detection of bacteria and eucarya having the same or similar nucleobase composition to the PNA probes claimed herein can be found in Table 3 of Amann et al. The table lists probes and nucleic acid sequences derived from relevant scientific literature.
Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide. a nucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide which can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes which are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc. 118:55 44-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay, appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
Though they hybridize to nucleic acid with sequence specificity (See: Egholm et al., Nature, at p. 567), PNAs have been slow to achieve commercial success at least partially due to cost, sequence specific properties/problems associated with solubility and self-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S., Tett. Lett. 36:6823-6826 (1995), Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P. E., Angew. Chem. Int. Ed. Engl. 35:1939-1942 (1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and Weiler, K., Nucleosides and Nucleotides 16:1775-1779 (1997) at p 433, col. 1, In. 28 through col. 2, In. 3) as well as the uncertainty pertaining to non-specific interactions which might occur in complex systems such as a cell (See: Good, L. et al., Antisense and Nucleic Acid Drug Development 7:431-437 (1997)). Consequently, their unique properties clearly demonstrate that PNA is not the equivalent of a nucleic acid in either structure or function. Thus, PNA probes need to be evaluated for performance and optimization to thereby confirm whether they can be used to specifically and reliably detect a particular nucleic acid target sequence, particularly when the target sequence exists in a complex sample such as a cell, tissue or organism.
PNA probes have been demonstrated as being useful for the detection of rRNA in ISH and FISH assays (See: WO95/32305 and WO97/18325). PNA probes have also been used in the analysis of mRNA (e.g. Kappa Light Chain), viral nucleic acid (e.g. human papillomavirus) and the analysis of centromeric sequences in human chromosomes and human telomeres (See: Lansdorp et al., Human Mol. Genetics, 5: 685-691 (1996) as well as WO97/14026). Similarly, the analysis of trinudeotide repeats in chromosomal DNA using appropriate PNA probes has been suggested (See: WO97/14026). A PNA probe has also been used to detect human X chromosome specific sequences in a PNA-FISH format (See: WO97/18325).
Any method, kits or compositions which could improve the specificity, sensitivity and reliability of probe-based assays for the detection of microorganisms in samples of interest would be a useful advance in the state of the art particularly where the methods were uniformly applicable to probes of all or substantially all sequence variations. Moreover, the methods, kits or compositions would be particularly useful if they could provide for the rapid, reliable and sensitive multiplex analysis of samples for the presence or absence of microorganisms and particularly bacteria and/or eucarya. The probes and assays would be particularly useful if they were well suited for the detecting, identifying or quantitating only colony forming units (viable organisms) in a sample.
This invention is directed to PNA probes, probe sets, methods and kits useful for the universal detection, identification and/or enumeration of bacteria and/or eucarya in a sample. The preferred probing nucleobase sequence of the universal probes of this invention are listed in Table 1, below. In preferred embodiments, PNA probes are organized into a set which is designed to detect, identify or quantitate the bacteria and/or eucarya which may be present in the sample. In a most preferred embodiment, the probe set is suitable for the specific detection, identification and/or quantitation of the total bacteria and/or total eucarya present in a sample.
Unique PNA probe constructs of this invention also include probes comprising two or more different types of labels such as the use of a hapten/fluorophore (e.g. fluorescein) in combination with an enzyme (e.g. soy bean peroxidase). Such orthogonally labeled probes can be experimental controls used in complex analysis systems or otherwise merely be used to provide alternative detection methodologies.
This invention is further directed to a method suitable for detecting, identifying or quantitating one or more bacteria and/or one or more eucarya in a sample. The method comprises contacting the sample with one or more PNA probes, wherein suitable probes are described herein. According to the method, bacteria and/or eucarya are then detected, identified or quantitated. Detection, identification and/or quantitation is made possible by correlating the hybridization, under suitable hybridization conditions, of the probing nucleobase sequence of a PNA probe to the target sequence of bacteria or eucarya in the sample to thereby determine the presence, absence or number of bacteria and/or eucarya in the sample. This correlation is made possible by direct or indirect detection of the probe/target sequence hybrid.
In yet another embodiment, this invention is also directed to kits suitable for performing an assay which detects, identifies or quantitates the bacteria and/or eucarya in a sample. The kits of this invention comprise one or more PNA probes (as described herein) and other reagents or compositions which are selected to perform an assay or otherwise simplify the performance of an assay.
The PNA probes, probe sets, methods and kits of this invention have been demonstrated to be specific and yet universal for either bacteria or eucarya by the Examples contained herein. By xe2x80x9cspecific and yet universalxe2x80x9d we mean that the PNA probes of this invention, either alone or in combination, detect target sequences within virtually all bacteria or eucarya, as the case may be, without any substantial cross reaction with non-target organisms. By xe2x80x9cvirtually all bacteria or eucaryaxe2x80x9d we mean that less than 2-4 percent of the target organisms will not be detectable with these universal probes (based on analysis of sequence information available in Genebank 104). By xe2x80x9cwithout substantial cross reactionxe2x80x9d we mean that less than 2-4 percent of non-target organisms will be detected when using these probes (based on analysis of sequence information available in Genebank 104). Moreover, the assays described herein are rapid (the entire assay can typically be performed in 3 hours or less with the probe hybridization requiring only 15-60 minutes), sensitive, reliable and generally applicable to probes of significantly different probing nucleobase sequence length and sequence variation. They can be used in an assay to accurately detect, identify and/or quantitate the total bacteria and/or total eucarya in a sample.
This invention is also directed to a multiplex PNA in-situ hybridization (PNA-ISH) assay and particularly a PNA-FISH assay. As a demonstration of the versatility of the PNA probes, probe sets, methods and kits of this invention, a fluorescent in-situ hybridization assay was multiplexed without any change to the protocol. The assay clearly provided for the detection, classification and/or enumeration of bacteria and eucarya in the sample (See: Example 12, herein). Thus, Applicants have demonstrated (believed to be the first successful example) the feasibility of a multiplex PNA-FISH assay which is suitable for, in a single assay, positively detecting, characterizing and quantitating the total bacteria and/or total eucarya present in a sample.
Since probe-based analysis detects nucleic acid without regard to the metabolic state of the organism in which the genetic material exists, the analysis of cells in culture is preferably used to distinguish between viable organisms and dead (non-viable) organisms, the presence of which are generally not considered to cause spoilage or contamination. Consequently, this invention is further directed to a rapid culture-based detection, identification and/or enumeration of total viable bacteria and/or total viable eucarya in a sample of interest.
The PNA probes, probe sets, methods and kits of this invention are particularly useful for the detection, identification and/or enumeration of bacteria and eucarya (e.g. pathogens) in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. The analysis of preferred non-limiting beverages include soda, bottled water, fruit juice, beer, wine or liquor products. Suitable PNA probes, probe sets, methods and kits will be particularly useful for the analysis of raw materials, equipment, products or processes used to manufacture or store food, beverages, water, pharmaceutical products, personal care products dairy products or environmental samples.
Additionally, the PNA probes, probe sets, methods and kits of this invention are particularly useful for the detection of bacteria and eucarya (e.g. pathogens) in clinical samples and clinical environments. Suitable PNA probes, probe sets, methods and kits will be particularly useful for the analysis of clinical specimens, equipment, fixtures or products used to treat humans or animals.